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Query: EC:2.7.7.6 (RNA polymerase)
 34,946 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

1. The surface of the RNA-polymerase-DNA complex possesses an exposed polypeptide loop. 2. Proteinases with differing specificities (trypsin, chymotrypsin, subtilisin and clostripain) preferentially cleave the exposed region. 3. The cleaved polypeptide is reassembled into RNA polymerase by renaturation from a solvent which promotes a random coil conformation. 4. Isolated beta subunit has a proteolytically resistant nucleus of approximately 70000 molecular weight. This resistant polypeptide may be generated by trypsin, chymotrypsin, subiilisin or clostripain. 5. Isolated alpha subunits are comparatively resistant to proteolysis. 6. Although of similar molecular weights beta and beta' appear to have unrelated primary sequences and markedly different conformations in free solution. 7. Digestion of the beta subunit may be blocked by formation of the alpha2beta subassembly. 8. Evidence is presented suggesting that beta' in the intact enzyme (alpha2beta beta') possesses the exposed polypeptide loop.
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PMID:Structural properties of Escherichia coli RNA polymerase Subunits. 77 11

The start point for transcription of the subtilisin (aprE) gene was determined by primer extension analysis and was found to be at a point significantly different from that identified in a previously published report (S. L. Wong, C. W. Price, D. S. Goldfarb, and R. H. Doi, Proc. Natl. Acad. Sci. USA 81:1184-1188, 1984). An aprE-lacZ fusion was used to analyze expression of the promoter. Deletion analyses of the promoter were performed to determine the extent of the upstream region necessary for activity. This was found to be between -52 and -41 with respect to the transcription start site. Expression of the aprE-lacZ fusion was unimpaired in a mutant deleted for the sigma B subunit of RNA polymerase. Mutations in the gene for the sigma H subunit of RNA polymerase decreased expression of the aprE-lacZ fusion to approximately 25% of that of the wild type. These results leave the identity of the sigma factor responsible for transcription of this gene in question. Mutations in the spo0A gene drastically decreased the activity of the aprE promoter and its upstream deletion derivatives, while the abrB gene, a phenotypic suppressor of spo0 mutations, restored activity of the aprE promoter in all of the deletion derivatives. Thus, inhibition of transcription by the spo0A mutation and its restoration by an abrB mutation could not be separated from the promoter of the aprE gene.
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PMID:Transcription of Bacillus subtilis subtilisin and expression of subtilisin in sporulation mutants. 244 62

In vitro studies demonstrated that the Bacillus subtilis subtilisin gene (aprE) could be transcribed by RNA polymerase holoenzyme reconstituted from core and sigma A factor obtained from vegetative cells. Upstream deletions (from -45) reduced the amount of transcription from the promoter. A deletion downstream of the promoter that overlapped a putative downstream minor promoter did not affect transcription from the sigma A promoter, which indicated that the putative downstream promoter is not utilized in vivo. S1 nuclease mapping studies showed that there was a low level of transcription from the subtilisin promoter during the growth phase and that the site of transcription initiation was the same during log and stationary phases. We conclude from these findings that there is only one promoter for the subtilisin gene and that it can be transcribed by the sigma A form of RNA polymerase in vitro.
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PMID:Bacillus subtilis subtilisin gene (aprE) is expressed from a sigma A (sigma 43) promoter in vitro and in vivo. 249 13

The monoclonal antibody (mAb) 64D1 was found to inhibit cAMP binding by the cAMP receptor protein (CRP) from Escherichia coli (Li, X.-M., and Krakow, J. S. (1985) J. Biol. Chem. 260, 4378-4383). CRP is relatively resistant to attack by the Staphylococcus aureus V8 protease, chymotrypsin, trypsin, and subtilisin whereas both mAb 64D1-CRP and cAMP-CRP are attacked by these proteases yielding N-terminal core fragments. The fragment patterns resulting from proteolysis of mAb 64D1-CRP and cAMP-CRP differ indicating that the CRP in each complex is in a different conformation. The data presented indicate that the preferred conformation of the antigenic site for mAb 64D1 is present in unliganded CRP. Binding of mAb 64D1 to CRP is inhibited at high cAMP concentration. Formation of a stable cAMP-CRP-lac P+-RNA polymerase open promoter complex resistant to dissociation by mAb 64D1 occurs at a much lower cAMP concentration. The observed increase in resistance to mAb 64D1 may reflect a possible conformational change in CRP effected by contact with RNA polymerase in the open promoter complex.
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PMID:A monoclonal antibody that inhibits cyclic AMP binding by the Escherichia coli cyclic AMP receptor protein. 303 13

The promoter region of Bacillus subtilis subtilisin E was found to be composed of two overlapping promoters with their transcription starting sites separated from each other by 15 base pairs (Wong, S.-L., Price, C. W., Goldfarb, D. S., and Doi, R. H. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1184-1188). At least one of the promoters is transcribed by a minor form of B. subtilis RNA polymerase with a sigma factor of 37,000 daltons. In vitro transcription analyses and in vivo studies with promoter probe plasmids pKO-1 and pCED-6 demonstrated that Escherichia coli RNA polymerase was able to initiate transcription from the subtilisin promoter cluster. S1 nuclease-mapping studies with both in vivo and in vitro transcribed RNA from E. coli and B. subtilis illustrate that E. coli can initiate transcription from both promoters with the same transcription start points as B. subtilis. The promoter strength of this promoter cluster in E. coli, as expressed in terms of galactokinase units, was 64 units and represents weak promoter activity in the E. coli system. These data indicate that either the single E. coli RNA polymerase is able to recognize the minor sigma 37 promoter or E. coli contains a hitherto unrecognized minor RNA polymerase holoenzyme which is capable of recognizing a B. subtilis sigma 37 promoter. On the other hand the B. subtilis RNA polymerase holoenzymes have been quite promoter-specific in our experiments to date.
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PMID:Utilization of a Bacillus subtilis sigma 37 promoter by Escherichia coli RNA polymerase in vivo. 608 47

A cloned Bacillus subtilis gene (sprE) expressed only during the stationary growth phase is shown to encode the subtilisin E protease, an enzyme associated with sporulation. We have determined the DNA sequence of the sprE promoter region and the promoter-proximal half of the structural gene. The sprE gene codes for a putative 29-residue signal peptide and a 77-residue leader peptide preceding the mature subtilisin sequence. By plasmid integration and phage PBS1 transduction, we have mapped the sprE locus between glyB and metD on the B. subtilis chromosome, a region also containing the hyperprotease-producing hpr gene. In vitro the sprE gene is transcribed by the minor form of RNA polymerase containing a 37,000-dalton sigma factor (sigma 37). We show by S1 nuclease mapping that sprE transcription initiates at dual start sites both in vitro and in vivo and that the promoter for the downstream site has a characteristic sigma 37 recognition sequence. We propose that the physiological role of the sigma 37 RNA polymerase is to transcribe a class of genes that are catabolite repressed, that encode extracellular enzymes, or that are expressed only during the stationary phase of growth.
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PMID:The subtilisin E gene of Bacillus subtilis is transcribed from a sigma 37 promoter in vivo. 632 90

The gene structure and expression of the Dfur2 gene of Drosophila melanogaster, which encodes the subtilisin-like serine endoprotease Dfurin2, was studied. The Dfur2 gene is very compact in contrast to the related Dfur1 gene, which has an estimated size of over 100 kbp. The 6-kb Dfur2 mRNA is encoded by 16 exons dispersed over a genomic region of about 9 kbp. The exon/intron organization shows conservation of intron positions not only in comparison with Dfur1, but also with the related mammalian genes FUR, PC1/PC3, PC2, and PC4. This conservation supports the hypothesis that all genes belonging to the family of subtilisin-like pro-protein processing enzymes are evolutionary related by descent from a common ancestral gene. In primer extension experiments, Dfur2 transcription initiation sites were identified in the presumed Dfur2 promoter region. This region was found to contain general RNA polymerase II promoter elements like a potential TATA box, a potential CAP signal, and several potential CCAAT boxes. Also, several sequence motifs putatively corresponding to binding sites for Drosophila transcription factors like zeste, bicoid, and engrailed were found to be present. RNA in situ hybridization experiments on Drosophila embryos revealed presumably maternal Dfur2 expression until the syncytial blastoderm (stage 5 of embryogenesis), no expression during gastrulation (stage 9), transient expression in a subset of neurons in the central nervous system of stage 12-13 embryos, and, from stage 13 onwards, expression in the developing tracheal tree. In a vaccinia expression system, the endoprotease Dfurin2 not only cleaved wild-type precursor of von Willebrand factor (pro-vWF) with pro-region cleavage site R-S-K-R decreases, but also, although to a lesser extent, pro-vWF mutants in which the P2 (vWFK-2A) or P4 (vWFR-4A) basic residue with respect to the pro-region cleavage site had been mutated. This cleavage specificity resembles that of human furin. The cleavage of pro-vWF by Dfurin2 shows that the previously reported lack of cleavage of the precursor of the beta A-chain of activin-A by Dfurin2 in this vaccinia expression system is substrate determined.
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PMID:The Dfur2 gene of Drosophila melanogaster: genetic organization, expression during embryogenesis, and pro-protein processing activity of its translational product Dfurin2. 788 Apr 43

The aprE gene of Bacillus subtilis encodes the major serine alkaline protease known as subtilisin. It is expressed during the transition state and transcribed by the sigma(A) form of the RNA polymerase (RNAP). In this work, we characterized the regulatory region of the aprE gene (rraprE) from B. subtilis. By computer analysis and site-directed mutagenesis, we localized the aprE promoter sequence 7 bp upstream from its transcription initiation site (TIS). We also characterized the static curvature properties of the rraprE DNA and found two different areas of DNA bending, within the first 400 bp upstream of its TIS. We postulate that these particular curved DNA regions could play a role in the interaction with some regulatory proteins and discuss possible implications related to aprE transcription regulation.
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PMID:Characterization of the 5' subtilisin (aprE) regulatory region from Bacillus subtilis. 1065 Jan 95

The structure and distribution of PC5-A, a prohormone convertase that is thought to be involved in post-translational processing of peptide hormone and neuropeptide precursors, have not been investigated in submammalian vertebrates. In the present study, we characterized the cDNA encoding PC5-A in the European green frog Rana esculenta. The frog PC5-A cDNA encodes a 913-amino acid protein that encompasses a 28-amino acid signal peptide, the Asp/His/Ser catalytic triad found in all serine proteinases of the subtilisin family, and two potential N-linked glycosylation sites located in a C-terminal cysteine-rich domain. Reverse transcriptase polymerase chain reaction amplification showed that PC5-A mRNA is expressed in various organs including the brain, spinal cord, pituitary, lung, liver, intestine, and testis, but not in the stomach and pancreas. The distribution of PC5-A mRNA in the frog brain was studied by in situ hybridization histochemistry. Intense expression was observed in the mitral cellular layer of the olfactory bulb, the nucleus of the diagonal band of Broca, the anterior preoptic area, and the suprachiasmatic and ventral hypothalamic nuclei. The expression pattern of PC5-A mRNA in the central nervous system of anuran amphibians was consistent with the implication of this prohormone convertase in the processing of various neuropeptide precursors.
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PMID:Molecular characterization of the cDNA and localization of the mRNA encoding the prohormone convertase PC5-A in the European green frog. 1250 14

The apicomplexan parasite Cryptosporidium is a significant cause of diarrheal disease worldwide. Previously, we reported that a Cryptosporidium parvum subtilisin-like serine protease activity with furin-type specificity cleaves gp40/15, a glycoprotein that is proteolytically processed into gp40 and gp15, which are implicated in mediating infection of host cells. Neither the enzyme(s) responsible for the protease activity in C. parvum lysates nor those that process gp40/15 are known. There are no furin or other proprotein convertase genes in the C. parvum genome. However, a gene encoding CpSUB1, a subtilisin-like serine protease, is present. In this study, we cloned the CpSUB1 genomic sequence and expressed and purified the recombinant prodomain. Reverse transcriptase PCR analysis of RNA from C. parvum-infected HCT-8 cells revealed that CpSUB1 is expressed throughout infection in vitro. In immunoblots, antiserum to the recombinant CpSUB1 prodomain revealed two major bands, of approximately 64 kDa and approximately 48 kDa, for C. parvum lysates and proteins "shed" during excystation. In immunofluorescence assays, the antiserum reacted with the apical region of sporozoites and merozoites. The recombinant prodomain inhibited protease activity and processing of recombinant gp40/15 by C. parvum lysates but not by furin. Since prodomains are often selective inhibitors of their cognate enzymes, these results suggest that CpSUB1 may be a likely candidate for the protease activity in C. parvum and for processing of gp40/15. Importantly, the recombinant prodomain inhibited C. parvum infection of HCT-8 cells. These studies indicate that CpSUB1 plays a significant role in infection of host cells by the parasite and suggest that this enzyme may serve as a target for intervention.
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PMID:Role of CpSUB1, a subtilisin-like protease, in Cryptosporidium parvum infection in vitro. 1916 60


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